The goal of the present research program is to utilize a genome-wide, high-throughput gene knock-out technology in combination with cellular assays to identify anti-inflammatory drug targets. This novel knockout technology, termed Genome-wide Cell-based Knockout technology or GECKO, is capable of inactivating expression of each transcriptionally active gene in cultured cells. Genome-wide GECKO libraries contain a relatively small number of clones, typically less than 1,000,000 clones, wherein each clone contains a different gene knockout, and collectively each transcriptionally active gene has been knocked out within the library. In this phase I research program, we will establish mast cell activation assays compatible with the GECKO technology, and create a genome-wide GECKO library for identification of genes whose function is necessary for mast cell degranulation. The specific aims of this phase I program, therefore, are 1) to develop robust high-throughput assays to enable screens for mast cell mutants with defective activation and degranulation responses.; and 2) to generate a phenotypically stable, genome-wide, GECKO library for use in identifying degranulation mutants in the chosen mast cell model. Success in this Phase I program will enable a full scale effort to identify genes that inhibit various mast cell functions involved in allergy, asthma, and anaphylaxis as well as in degenerative fibrosis and arthritis, and thereby identify new drug targets for the treatment of inflammatory diseases.